Type II thioesterase from streptomyces coelicolor a3(2) and the coding sequence

ABSTRACT

The invention concerns a biosynthesis of nonaramatic polyketide compounds. More particularly, the present invention relates to a isolated polynucleotide molecule, a nucleic acid vector comprising the molecule, and isolated polypeptide and uses thereof. A new, type II thioestrerase can be obtained as an application outcome of this invention. The enzyme is active in polyketide, especially macrolide, biosynthesis when associated with a multienzyme complex of a polyketide synthase. The activity of the enzyme results in a increase of polyketide biosynthesis efficiency.

The present invention relates generally to the fields of molecularbiology. More particularly, it concerns a biosynthesis of nonaromaticpolyketide compounds.

More particularly, the present invention relates to a isolatedpolynucleotide molecule, a nucleic acid vector comprising the molecule,an isolated polypeptide and uses thereof. A new, type II thioestrerasecan be obtained as an application outcome of this invention. The enzymeis active in polyketide, especially macrolide, biosynthesis whenassociated with a multienzyme complex of a polyketide synthase. Theactivity of the enzyme results in an increase of polyketide biosynthesisefficiency.

Polyketides are a large and structurally a diverse group of compoundssynthesized by microorganisms (bacteria and fungi) and by plants. Thecompounds are synthesized as a result of multiple, small carboxylic acidcondensation and reduction cycles. Most of the known, naturallyoccurring polyketides, are produced by soil bacteria, streptomycetes.Polyketides are known as biologically active compounds, most of whichare commonly used antibiotics, immunomodulators and anticancer drugs.

Nascent polyketides are processed by large multienzyme complexes,polyketide synthases (PKS). In the type I PKS, involved in production ofmacrolide antibiotics such as erythromycin, reactions of thebiosynthetic cycle are catalysed sequentially by separate enzymicdomains housed in large multifunctional polypeptides. Each completecycle of condensation and reduction reactions is catalysed by a module,a functional unit of the PKS. The substrate acyl chains which undergosuccessive reactions are tethered as thioesters by acyl carrier domainsof the PKS polypeptides. A terminal thioesterase domain (TE) catalysesrelease and cyclization of the full-length (fully processed) polyketidechain (Katz & Donadio, 1993).

Many type I PKS, and also nonribosomal peptide synthetase (NRPS)clusters, contain additional TE genes located adjacent to the PKS geneswithin the cluster of antibiotic biosynthetic genes (Weissman et al.,1998; Schneider & Marahiel, 1998; Shaw-Reid et al., 1999; August et al.,1998; Xue et a!., 2000; Heathcote et al., 2001). The products of suchgenes are discrete proteins called type II thioesterases (TE II) todistinguish them from chain-terminating thioesterase domains, TE I(Gokhale et al., 1999).

The function of type II thioesterases is predicted from gene disruptionanalysis, complementation studies, and determination of their substratespecificities (Weissman et al., 1998; Butler et al., 1999; Heathcote etal., 2001). Polyketide production is drastically reduced, by 90% ormore, in strains with deleted TE II gene (Xue et al., 2000; Butler etal., 1999; Doi-Katayama et al., 2000), indicating an important function,proposed to involve editing of aberrant intermediates (Butler et al.,1999) during the course of polyketide biosynthesis. More recently, thishypothesis has been confirmed and the mechanism clarified. Thus, theTylO protein displayed hydrolytic activity in vitro towards short chainacyl-CoAs, indicating that the enzyme could remove aberrantlydecarboxylated (and, therefore, non-reactive) extender acyl chains fromthe PKS during polyketide biosynthesis (Heathcote et al., 2001). Byhydrolytic release of such aberrant acyl groups, TE II was proposed tounblock PKS modules and restore overall efficiency of the complexenzyme.

Modular polyketide synthases, as demonstrated by numerous experiments,can serve as efficient tools for the combinatorial biosynthesis of newpolyketides. The latter include both analogues of existing polyketidesand compounds with a novel activity. Genetic engineering studies allowassembly of novel polyketide chains following fusion, swapping orrepositioning of catalytic domains, modules or whole peptides within PKSpolypeptides (Hutchinson & Fujii, 1995; Ranganathan et al., 1999; Tanget al., 1999). In engineered PKSs, co-expression of TE IIs in additionto other PKS proteins, might help in achieving elevated levels of thepolyketide products.

This invention is directed towards production of a new, type IIthioesterase as an expression product of the new gene. The activity ofthe enzyme might play a beneficial role in nonaromatic polyketidesynthesis.

The subject of this invention is an isolated polynucleotide comprisingthe sequence having at least 60% homology to the nucleic acid sequenceof SEQ ID NO. 1 or its complementary strand or fragments thereof.Preferably, the polynucleotide comprises the sequence having at least70% homology to the nucleic acid sequence of SEQ ID NO:1 or itscomplementary strand or fragments thereof encoding TE II protein. Morepreferably, the polynucleotide comprises the sequence having at least80%, and more preferably at least 90%, homology to the nucleic acidsequence of SEQ ID NO:1 or its complementary strand or fragmentsthereof. Preferably the polynucleotide encodes tioesterase type IIproteine. Preferably the polynucleotide according to the inventionencodes at least 15, and more preferably at least 150, contiguous aminoacids from SEQ ID NO:2. Preferably, the polynucleotide encodespolypeptide comprising the amino acid sequence of SEQ ID NO:2.Preferably the polynucleotide comprises the sequence of SEQ ID NO:1 orSEQ ID NO:3 or complementary strand thereof.

In accordance to the invention, the DNA can be used for the expressionof TE II, both as a coding and a regulatory element. The DNA can be usedto obtain antisense strand and to design hybridization probes andprimers.

The other subject of the invention is any nucleic acid vector comprisingthe polynucleotide according to the invention, as defined above. Thevector can contain e.g. the entire sequence SEQ ID No. 1, a functionalequivalent or a specified element thereof, like the sequence encoding TEII protein, a fragment of the protein or the regulatory element.

The subject of the invention is also a protein or fragment thereofencoded by a polynucleotide according according to the invention, asdefined above.

The subject of the invention is also an application of a polynucleotideaccording to invention for the expression of a protein, preferably theprotein of TE II activity.

Applications of a polypeptide of an amino acid sequence essentiallyconsistent with SEQ ID No. 2 or fragments thereof for polyketidesynthesis are also addressed by the invention. The polypeptide can beused to obtain multienzyme polyketide synthases and/or to increasebiosynthetic efficiency of polyketide synthase complexes.

By the use of the methods similar to the ones disclosed in the examplesgiven below, the TE II gene from the chromosome of Streptomycescoelicolor A3(2) disclosed in this invention can be transferred to thechromosome of another streptomycete strain which produces a polyketide(possibly an antibiotic). Based on the disclosed results, one can expectthat the transferred gene will be expressed and a functional protein ofthe TE II activity will be produced. This would increase the efficiencyof the polyketide production. Thus the protein can be used forpolyketide synthesis. Especially, the protein can be used to obtain newmultienzyme polyketide synthases and to increase a biosynthesisperformed by known polyketide synthase complexes. A relatively broadsubstrate tolerance exhibited by the enzyme would help to apply the TEII in different, heterologous synthase complexes.

EXAMPLE 1 Isolation and Structure of the Gene scoT, Encoding a New TE II

Hybridization experiment was done by using DNA isolated from cosmidclones from the geneomic DNA library of the streptomycete—Streptomycescoelicolor A3(2) M145. Nonradioactively-labeled DNA, obtained as a PCRreaction product, was used as a probe. The substrate for nonradioactivelabeling in a course of random priming reaction (Sambrook et al., 1989)was digoxygenin-11-dUTP. Probe labeling, hybridization and hybriddetection was done according the procedure of Boehringer Mannheim (TheDIG System User's Guide for Filter Hybridization). The amino acidsequence of the probe was equivalent to the C-terminal region ofketosynthase domains from 6-deoxyerythronolide B synthase ofSaccharopolyspora erythraea (synthesizing an aglycone part oferythromycin A). Based on a hybridization with the probe, a cosmid clonewas chosen for further studies. In a course of these studies therestriction map of the cosmid was obtained. Another DNA probe of asequence comprising a central part of acyltransferase domain from type Ipolyketide synthase, including active site region, was used. Similar tothe former probe, the further one was a digoxygenin-labeled, PCRreaction product. The DNA reacting with the further probe, obtained astwo restriction fragments, was cloned in pBlueScript SK vector(Stratagene) in E. coli XL1Blue strain (Stratagene) by using theprocedures of Sambrook et al., (1989). The fragments were further clonedinto the same vector as a one, continuous insert.

DNA sequence of the cloned restriction fragments was obtained. Thesequence of S. coelicolor DNA was determined both manually (Kuczek etal., 1998) by using the chain-termination method with the Sequenase v.2.0 sequencing kit of Amersham and by automated sequencing performed byQiagen Sequencing Services, Hilden, Germany. The sequence was determinedon both strands and submitted to the GenBank database (AF109727). Thesequence AF109727, originally deposited in the database, containednumerous frame-shift mistakes which were later corrected and thecorrected sequence (SEQ ID No.1) was analysed. Computer-assistedsequence analysis, with the aid of CODONPREFERENCE program, revealed anopen reading frame of 807 bp, located on the two fragments. The ORFdesignated scoT (SEQ ID No.3) was deduced to code for a protein of 268amino acid residues (molecular mass 28 686 Da, isoelectric pH 6.17) ofwhich about 53% were predicted to be hydrophobic (SEQ ID No.2).

Comparison of the scoT sequence with others in the databases revealedextensive similarities with thiosterase enzymes from variousactinomycetes and other bacteria and also rat S-acyl fatty acid synthasecomplex. The greatest similarity was found with type II thioesterases(Pfam00975), namely, 43% identity with a TE II (AF040570) from therifamycin biosynthetic gene cluster of Amycolatopsis mediterranei, 43%identity with a TE II (X60379) associated with DEBS(6-deoxyerythronolide B synthase) from Saccharopolyspora erythraea, and40% identity with TylO (U08223), the TE II involved in tylosinbiosynthesis in Streptomyces fradiae. ScoT (SEQ ID No.2) is predicted tobelong to the well-known alpha/beta hydrolase family (Pfam00975).Comparisons of the nucleotide and amino acid sequences with thedatabases were performed with the BLAST and ClustalW programs.

Comparison of the SEQ ID No.1 sequence with others in the databases alsorevealed that the sequence comprises a region active in atranscriptional regulation of scoT.

EXAMPLE 2 Expression of scoT and Confirmation of ScoT Protein Activity:Complementation of a Knockout Mutant of the S. fradiae Natural TE IIGene by the TE II Gene of S. coelicolor A3(2)

We attempted to determine whether the product of scoT has TE IIactivity. This was examined by studying whether it could functionallyreplace the native TE II (i.e. TylO) in the tylosin producer,Streptomyces fradiae. ScoT and TylO show extensive amino acid sequencesimilarity and the complementation system for TE II gene-disrupted S.fradiae strain was constructed (Butler et al., 1999). Therefore, scoTwas cloned in a conjugative expression vector pLST9828 and integratedupon transconjugation from E. coli into the chromosome of atylO-disrupted strain of S. fradiae. The vector was constructed in thelaboratory of Prof. E. Cundliffe (University of Leicester, Leicester,U.K.).

There are two unique restriction sites suitable for cloning in pLST9828:BamHI and XbaI. As BamHI site is also present within the scoT sequence,the gene was ligated into pLST9828 in a two-step process. First, the DNAcontaining the C-terminal part of ScoT was PCR amplified from thepBSK(−) plasmid clone template using M13 reverse primer and the TE-Revprimer with an engineered XbaI site (underlined):5′-TTTTCTAGATGTCGTACGTACACGGA-3′. The PCR product was purified using theQiaex II DNA purification kit, digested and ligated into pLST9828 usingthe BamHI and xbaI sites. Then, a second PCR product containing theN-terminal part of ScoT was obtained from the template of anotherpBSK(+) construct using the T7 universal primer and the TE-Fw primerwith an engineered BanHI site (underlined):5′-TTTTTTGGATCCGATGGGAAGTGACTGGTT-3′. The 50 μl reaction mixturecontained 5 μl of 10×PCR DyNAzyme buffer (Finnzymes), 1 μl of 10 mMdeoxynucleoside triphosphate mixture, 50 pmol of each oligonucleotide,about 10 ng of template DNA and 1 μl of DyNAzyme™ II DNA polymerase(Finnzymes). Cycling was as follows: hot start at 96° C. for 6 min, 1min at 80° C. (adding of the enzyme), 31 cycles with denaturation at 95°C. for 1 min, annealing at 63-65° C. for 1 min and extension at 72° C.for 1.5 min, followed by a final extension at 72° C. for 5 min. Theproduct was digested with BamHI and ligated into the pLST9828 derivativeobtained in the first step of the cloning procedure. Gentamycin (15 μgml⁻¹) was used for selection of E. coli DH5α transformants. Theauthenticity and orientation of the cloned fragments was confirmed byautomated sequence analysis.

The gene cloned in pLST9828 was introduced, by transconjugation from E.coli S17-1 (Kieser et al., 2000), into S. fradiae strain, a disruptionmutant of the native TE II gene, tylO. Due to a polar effect of thedisruption of tylO on the expression of the downstream gene, thetylO-disrupted strain produce demycarosyl-tylosin (desmycosin) as dostrains in which the disruption is successfully complemented by clonedDNA (Butler et al., 1999).

Complemented mutant strains of S. fradiae were fermented andfermentation products were extracted and analysed by reverse phase HPLC,with absorbance measurement at 282 nm (Butler et al., 1999). Desmycosinwas used as an internal standard to identify fermentation products incomplemented strains. Transconjugation, as well as fermentation andproduct analyses were done in the laboratory of Prof. E. Cundliffe.

Desmycosin production was restored up to 48% of the level of macrolideproduced by the wild type strain. Control fermentation of thenon-complemented, tylO-disrupted strain yielded only minimal amounts ofdesmycosin. These results showed that the TE II gene, scoT, fromStreptomyces coelicolor A3(2) could, by complementation, restore to asignificant level macrolide production in the tylO-disrupted strain ofStreptomyces fradiae. The two enzymes appear, therefore, to beequivalent in their catalytic function.

Literature

-   August, P. R., Tang, L., Yoon, Y. J., Ning, S., Muller, R., Yu, T.    W., Taylor, M., Hoffmann, D., Kim, C. G., Zhang, X.,    Hutchinson, C. R. & Floss, H. G. (1998) Chem. Biol., 5, 69-79.-   Butler A. R., Bate N., Cundliffe E. (1999) Chem. Biol. 6: 287-292-   Doi-Katayama Y., Yoon Y. J., Choi Ch. Y., Yu T. W., Floss H. G.,    Hutchinson C. R. (2000) J. Antibiotics 53, 484-495.-   Gokhale R. S., Hunziker D., Cane D. E., Khosla Ch. (1999) J. Chem.    Biol. 6: 117-125-   Heathcote M. L., Staunton J., Leadlay P. F. (2001) Chem. Biol. 8:    207-220-   Hutchinson C. R., Fujii I. (1995) Annu. Rev. Microbiol 49: 201-238-   Katz L., Donadio S. (1993) Annu. Rev. Microbiol. 47: 875-912-   Kuczek, K., Kotowska, M., Wiernik, D. & Mordarski, M. (1998)    BioTechniques 24, 214-215.-   Tang L., Fu H., McDaniel R. (2000) Chem. Biol. 7: 77-84-   Xue Y, Wilson D., Sherman D. H. (2000) Gene 245: 203-211 Antibiot.    53: 484-495-   Kieser T., Bibb M. J., Buttner M. J., Chater K. F.,    Hopwood D. A. (2000) Practical Streptomyces Genetics, The John Innes    Foundation, Norwich-   Ranganathan, A., Timoney, M., Bycroft, M., Cortés, J., Thomas, I.    P., Wilkinson, B., Kellenberger, L., Hanefeld, U., Galloway, I. S.,    Staunton, J. & Leadlay, P. F. (1999) Chemistry & Biology 6, 731-741.-   Sambrook J., Fritsch E. F., Maniatis T. (1989) Molecular Cloning: a    Laboratory Manual, Cold Spring Harbor Laboratory Press-   Schneider, A. & Marahiel, M. A. (1998) Arch. Microbiol., 169,    404-410.-   Shaw-Reid, C. A., Kelleher, N. L., Losey, H. C., Gehring, A. M.,    Berg, Ch. & Walsh, Ch. T. (1999) Chemistry & Biology 6, 385-400.-   Weissman, K. J., Cameron, J. S., Hanefeld, U., Aggarwal, R.,    Bycroft, M., Staunton, J. & Leadlay, P. F. (1998) Chem. Int. Ed.,    37, 1437-1440.-   Xue, Y., Wilson, D. & Sherman, D. H. (2000) Gene 245, 203-211.

1. An isolated polynucleotide comprising the sequence having at least60% homology to the nucleic acid sequence of SEQ ID NO. 1 or itscomplementary strand or fragments thereof.
 2. The polynucleotide ofclaim 1, comprising the sequence having at least 70% homology to thenucleic acid sequence of SEQ ID NO:1 or its complementary strand orfragments thereof.
 3. The polynucleotide of claim 1, comprising thesequence having at least 80% homology to the nucleic acid sequence ofSEQ ID NO:1 or its complementary strand or fragments thereof.
 4. Thepolynucleotide of claim 1, comprising the sequence having at least 90%homology to the nucleic acid sequence of SEQ ID NO:1 or itscomplementary strand or fragments thereof.
 5. The polynucleotideaccording to one of claim 1-4, characterised in that encodes tioesterasetype II proteine.
 6. The polynucleotide of claim 1, wherein saidpolynucleotide encodes at least 15 contiguous amino acids from SEQ IDNO:2.
 7. The polynucleotide of claim 1, wherein said polynucleotideencodes at least 50 contiguous amino acids from SEQ ID NO:2.
 8. Thepolynucleotide of claim 1, wherein said polynucleotide encodes at least150 contiguous amino acids from SEQ ID NO:2.
 9. The polynucleotide ofclaim 1, wherein said polynucleotide encodes polypeptide comprising theamino acid sequence of SEQ ID NO:2.
 10. The polynucleotide of claim 1 or9, comprising the sequence of SEQ ID NO:1 or SEQ ID NO:3 orcomplementary strand thereof.
 11. A nucleic acid vector comprising thepolynucleotide according to anyone of claims 1 to
 9. 12. A protein orfragment thereof encoded by a polynucleotide according to anyone ofclaims 1 to
 10. 13. A use of a polynucleotide according to anyone ofclaims 1 to 10 for an expression of a protein.
 14. The use according toclaim 13 wherein the protein have the TE II activity.
 15. A use of apolypeptide comprising an amino acid sequence essentially consistentwith the sequence SEQ ID No. 2 or fragments thereof for polyketidesynthesis.
 16. The use according to claim 15 characterized in, that theprotein is used in a multienzyme polyketide synthase assembly.
 17. Theuse according to claim 15 characterized in, that the protein is used inorder to increase biosynthetic efficiency of a polyketide synthasecomplex.